The alpha-actinins are actin-binding proteins that include both cytoskeletal isoforms that organize actin filaments, and sarcomeric isoforms that represent major structural components of muscle Z-lines. Based on their interactions with many structural and signaling proteins associated with the Z-line and thin filament, sarcomeric alpha-actinins likely perform a static function in skeletal muscle to maintain the ordered myofibrillar array, as well as possible regulatory functions in coordinating myofiber structure and function. In humans, two genes encode the skeletal muscle alpha-actinins, ACTN2 and ACTN3. alpha-Actinin-2 is present in all skeletal muscle fibers, whereas alpha-actinin-3 is expressed only in fast (type 2) fibers. We have previously shown that alpha- actinin-3 deficiency is common in the general population due to homozygosity for a premature stop codon in ACTN3 (R577X). Absence of alpha-actinin-3 is not associated with a disease phenotype and it is likely that alpha- actinin-2 is able to "compensate" for the absence of alpha-actinin-3 in humans. Nevertheless, there must be subtle differences in isoform function as we have found that specialist sprint athletes have an extremely low frequency of the 577XX (alpha-actinin-3 null) genotype compared to controls, (p<0.0001), whereas elite endurance athletes have a high frequency of 577XX. We have now shown that transcriptional knockdown of alpha-actinin -2, but not alpha-actinin -3, results in changes in fiber type gene expression, and that knockout of alpha-actinin-2, but not -3, is embryonic lethal in mice. The central working hypothesis that we propose to test here is that the various alpha-actinin isoforms have evolved subtle differences in structure that allow them to perform distinctly different functions. This application proposes to utilize transcriptional knockdown and rescue experiments in cell culture and zebrafish developmental models, as well as transgenic and knockout mice, to study the differential functions of alpha-actinin-2 and -3 and their contributions to the unique physiological roles that fast and slow muscles perform. The experiments described in this proposal will make a significant contribution to our understanding of the differential roles of alpha-actinin isoforms in normal skeletal muscle biology, and the mechanism(s) by which these may influence normal human variations in fitness and performance. Our results may also lead to insights into the role(s) that alpha-actinin genotypes may play in modulating and/or causing human neuromuscular disease.